Two basic types of mutation assays have heretofore been employed which are reverse mutation assays and forward mutation assays.
A reverse mutation assay utilizes a mutant population that is deficient in some function, usually one involved in the synthesis of an essential cellular component. This deficiency results from an altered sequence of base pairs in the genetic material, DNA. This mutation renders the organism unable to grow unless supplemented with the missing component. The mutagenicity of a compound is defined by its ability to mutate the original DNA lesion back to the wild-type sequence or to suppress it by a second site mutation. The occurrence of such events can be detected by placing the treated population under selective (unsupplemented) conditions; only the revertants will grow and be detected. However, studies of the various molecular mechanisms of mutations have revealed that use of single reversion assays are not an acceptable method for screening chemical mutagens because particular mutagens cannot cause all types of mutations. Thus, sets of reversion assays are employed to reduce the probability of missing the activity of a mutagen.
In a forward mutation assay, a population of cells is treated with a suspected mutagen and then exposed to a selective agent which is toxic to wild-type cells. Mutants which lack the cellular function necessary for the toxic effect of the selective agent, such as an enzyme, can survive and be detected.
Assays for mutagenesis, both reverse and forward, have often been based upon bacterial cells or mammalian cells such as rodent cells. For example, McCann et al. have developed an assay employing S. typhimurium histidine reversion whereas Clive et al. have developed a forward mutation assay in L.sub.517 8Y mouse lymphoma cells using the thymidine analog bromodeoxyuridine as the selective agent. See McCann, J., Choi, E., Yamasaki, E. and Ames, B. N. (1975) Proc. Natl. Acad. Sci., 72: 5135; Clive, D. and Spector, J. F. S. (1975) Mutat. Res, 31, 17-29; Clive, D., Flamm, W. G. Machesko, M. R., and Bernheim, N. J. (1972) Mutat. Res, 16, 77-87; and Clive, D., Flamm, W. G., and Patterson, J. B. (1973) Chemical Mutagens: Principles and Methods for their Detections, Vol. 3, pp. 79-103, Plenum Publishing Co., New York. There are, however, important species differences in DNA/chromatin structure, DNA repair, membrane structure, and metabolism between human cells and bacterial and/or other mammalian cells.
One recently developed assay for mutagenesis is a forward mutation assay involving human diploid lymphoblast cells. This assay detects mutagenic damage at the hypoxanthine guanine phosphoribosyl transferase gene locus based upon the resistance of mutant lymphoblasts to purine analogs, such as 6-thioguanine, which serves as a substrate for this enzyme and is toxic to non-mutant lymphoblasts. While this assay appears to be quite promising, it does involve a relatively long phenotypic expression time (10-12 generations) before the resistance to the purine analog is completed. A complete description of this assay is presented in U.S. Pat. No. 4,066,510 issued to William G. Thilly.